Methods for Treating a Neoplastic Disease in a Subject Using Inorganic Selenium-Containing Compounds

ABSTRACT

The invention features methods and selenium-containing compositions for treating a neoplastic disease in a subject. In particular, the invention features methods for enhancing sensitivity of a tumor to cancer therapy by treating the tumor with an inorganic selenium-containing (iSe) compound and with a cancer therapy, particularly a cancer therapy that also affects the cellular redox status of a tumor cell (e.g., radiation).

FIELD OF THE INVENTION

The present invention is in the field of cancer therapy.

BACKGROUND OF THE INVENTION

Although cancer therapies have advanced greatly over the years,significant challenges remain. Cancer therapies are generally associatedwith undesirable side effects, highlighting the need for therapies thatare selective for tumor cells, and thus have decreased toxicity. Inaddition, chemotherapy- and radiation resistant cancers have a very highhurdle to successful therapy. In some instances, this resistance is dueto the resistance of the cancerous cell to apoptosis.

For example, in prostate cancer, the resistance of prostate cancer cellsto apoptosis plays a role in local and distant disease progressionfollowing conventional therapy (e.g. hormonal ablation andradiotherapy). The durable and local control rate (determined by serumlevels of prostate specific antigen (PSA)) for patients with prostaticcancers of various stages and grades treated with primary radiationtherapy alone is approximately 38%, and treatment of metastatic diseaseis palliative at best. The apoptotic machinery of most prostate cancercells is intact, however, due to molecular alterations the cells areunable to execute the apoptotic pathways.

Selenium, a key component of a number of functional selenoproteinsrequired for normal health, when in the inorganic selenite or an organicform such as selenomethione, has been shown to have both preventive andtherapeutic effects. Inorganic and organic selenite can inhibittumorigenesis in a variety of animal models at doses in excess of thoserequired to support maximal activity of selenoproteins (Ip, et al.,Current concepts of selenium and mammary tumorigenesis, In: Cellular andMolecular Biology of Breast Cancer, 479-494. Plenum Press, N.Y. (1997);Medina et al., Pathol Immunopathol Res, 7: 187-199 (1988); Milner etal., Fed Proc, 44: 2568-2572 (1985)). Epidemiology studies have shown astatistically significant inverse relationship between selenium levelsand cancer risk (Combs et al., Selenium and cancer, In: Antioxidants andDisease Prevention, Ch. 8, 97-113. CRC Press, N.Y. (1997); Shamberger etal., CRC Crit Rev Clin Sci, 2: 211-219 (1971)). Human cancer preventiontrials have shown that daily oral supplementation with ofselenium-enriched yeast containing mostly L-selenomethionine (200μg/day, approximately four times the recommended daily value) cansignificantly reduce the incidence of several major cancers includingprostate, colon, and lung by nearly 50% (Clark et al., JAMA, 276:1957-1963 (1996)).

While the majority of selenium research has focused on the use oflong-term selenium intake for chemoprevention, little attention has beengiven to the cytotoxic effects of selenium and the potential use ofselenium for chemotherapy in the clinical setting. The anti-tumoractivities of selenium compounds are dependent upon the dose andchemical form. Selenite (oxidation state +4) undergoes thiol-dependentreduction to selenide (H₂Se), which supplies selenium for the synthesisof selenoproteins, whereas selenomethionine is converted toselenocysteine before being degraded by the enzyme β-lyase to H₂Se(Combs et al., Pharmacol. Ther., 79(3): 179-192 (1998)). Selenitemetabolism results in the generation of superoxide and oxidative stressthrough its reductive reaction with reduced GSH (FIG. 1) (Combs, 1998).Selenate is metabolized to selenite in the body.

Selenite is capable of inhibiting cell growth and inducing apoptosis ina variety of human cancer cells lines in vitro (Menter et al., CancerEpid Bio Prev, 9: 1171-1182 (2000); Zhong et al., Cancer Res, 61:7071-7078 (2001)). Selenite (2 mg/kg, subcutaneous injection) has alsobeen shown to inhibit the tumor growth of breast and ovarian cancer celllines in vivo without apparent ill effects on the host (Watrach et al.,Cancer Letters, 25: 41-47 (1984); Watrach et al., Cancer Letters, 15:137-143 (1982); Caffrey et al., Cancer Letters, 121: 177-180 (1997)).The induction of apoptosis by Selenite is mediated by a redox mechanisminvolving induction of oxidative stress via superoxide formation andlowered intracellular GSH levels (Zhong, 2001). Mitochondria appear toserve as the main target for Selenite-induced apoptosis, with subsequentrelease of cytochrome c, followed by mitochondrial depolarization,caspase-3 activation and DNA fragmentation (Shen et al., Free Rad BiolMed, 30(1): 9-21 (2001). Several studies have also reported thatselenium compounds selectively induce growth inhibition and apoptosis incancer cells compared to normal cells (Menter, 2000; Fleming et al., NutCancer, 40(1): 42-49 (2001); Ghose et al., Cancer Res, 61: 7479-7487(2001)). However, the molecular pathways underlying the differentialresponse are poorly understood.

Thus, there remains a need in the field for methods of treatingneoplastic disease, particularly drug- and radiation-resistantneoplasms, and particularly for improving the sensitivity of tumors tocancer therapy. The present invention addresses these needs.

LITERATURE

Micke et al., Int. J. Radiation Oncology Biol. Phys., 56(1):44-49(2003); Frankel at al., Curr Pharm 7(16):1595-614 (2001); Caffrey etal., Cancer Chemother. Pharmacol. 46(1):74-8 (2000); Menter et al.,Cancer Epid. Biomarkers. Prev., 9:1171-1182 (2000); Buntzel, Med. Klin.,3:49-53, 94 Suppl. (1999); Caffrey et al., Biol. Trace. Elem. Res.65(3):187-98 (1998); Caffrey et al., Cancer Lett. 121(2):177-80 (1997);Baldew et al., Cancer Res., 49(11):3020-3 (1989); Ohkawa et al., Br. J.Cancer, 58(1):38-41 (1988); Webber et al., Biochem. Biophys. Res.Commun., 130(2):603-609 (1985); Shen et al., Int. J. Cancer. 81: 820-828(1999); Leung et al. Cancer. 71: 2276-85 (1993); Perry et al. Cancer.72(3): 783-787 (1993); Cook et al. Cancer Res. 51: 4287-4294 (1991);Mackey et al. Urology. 52(6): 1085-1090 (1998); Gleave et al. Clin.Cancer Res. 5: 2891-8 (1999); Gleave et al. Urology. 54(6A): 36-46(1999); Shen et al. Free Radic. Biol. Med. 30(1): 9-21 (2001); Menter etal. Cancer Epidemiol. Biomarkers Prev. 9: 1171-1182 (2000); Zhong et al.Cancer Res. 61: 7071-7078 (2001); Jiang et al. Mol. Cancer Ther. 1:1059-1066 (2002); Ghosh et al. Biochem Biophys. Res. Comm 315: 624-635(2004); Watrach et al. Cancer Lett. 25: 41-47 (1984); Zhou et al. J.Biol. Chem. 278(32): 29532-29537 (2003); Shen et al. Free Radic. Biol.Med. 28(7):1115-1124, 2000; Greeder et al. Science. 209: 825-827 (1980);Watrach et al. Cancer Lett. 15: 137-143 (1982); Milner et al. CancerRes. 41: 1652-1656 (1981); Caffrey et al. Cancer Lett. 121: 177-180(1997); Corcoran et al. J. Urol. 171: 907-910 (2004).

SUMMARY OF THE INVENTION

The invention features methods and inorganic selenium-containingcompositions for treating a neoplastic disease in a subject. Inparticular, the invention features methods for enhancing sensitivity ofa tumor to cancer therapy by treating the tumor with an inorganicselenium-containing (iSe) compound and with a cancer therapy,particularly a cancer therapy that also affects the cellular redoxstatus of a tumor cell (e.g., radiation).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing how selenite metabolism results in thegeneration of superoxide and oxidative stress through its reductivereaction with reduced GSH.

FIG. 2 shows the results of analysis of selenite-induced apoptosis inLAPC-4 prostate cancer cells. Panel A: Cells were treated with seleniteat the indicated concentrations for 48 hours (closed squares, selenitealone; open squares, pre-treatment with buthionine sulfoximine (BSO)(500 μM) for 24 hours). Percent apoptosis was detected as the fractionof cells in sub-G₁ by FACS analysis of PI stained cells. Panel B:Caspase 3 cleavage in LAPC-4 cells following exposure to selenite asdetected by Western blot analysis. Panels C and D Ratio of GSH:GSSG(reduced glutathione to glutathione disulfide) and total GSHconcentrations in LAPC-4 cells following 48 hour selenite treatmentmeasured using the GSH-reductase recycling assay. Values represent themean±SD for 3 individual experiments.

FIG. 3 shows results of analysis of selenite-induced apoptosis inmatched pairs of normal prostate and prostate cancer primary cellstrains. Cells were treated with selenite for 48 hours and apoptosis wasdetected as the fraction of cells in sub-G₁. Closed squares, normalprostate (PZ); open squares, prostate cancer (CA). Panels A, B, and C:Effects of selenite on matched pairs E-PZ/CA-1, E-PZ/CA-2, and E-PZ/CA-3respectively. Panel D: Representative examples of cell cycle profiles ofE-PZ/CA-3 cells and sub-G1 determinations following selenite treatment.Values represent the mean±SD for 3 individual experiments.

FIG. 4 shows results of analysis of intracellular GSH content in primaryprostate (normal and cancer) cell strains. Panel A: The ratios ofGSH:GSSG; Panel B: total GSH concentrations, and Panel C GSSGconcentrations in E-PZ/CA-1, E-PZ/CA-2, and E-PZ/CA-3 cells measuredusing the GSH-reductase recycling assay. Closed squares, normal prostate(PZ); open squares, prostate cancer (CA). Values represent the mean±SDfor 3 individual experiments.

FIG. 5 shows results of analysis of the effects of selenite on theintracellular GSH:GSSG ratio in E-PZ/CA-3 cells. Panel A: Cells weretreated with selenite for 48 hours and the ratio of GSH:GSSG wasmeasured using the GSH-reductase recycling assay. Closed squares, normalprostate (PZ); open squares, prostate cancer (CA). Panel B: Percentreduction in the GSH:GSSG ratio following treatment with selenitecompared to untreated levels. Values represent the mean±SD for 3individual experiments.

FIG. 6 shows the effects of selenite on intracellular GSH content inLAPC-4 cells. Cells were treated with 10 μM selenite for 6, 24, and 48hours and total GSH and GSSG concentrations were measured using theGSH-reductase recycling assay. Changes in Panel A, total GSH, Panel B,GSSG, and Panel C, GSH:GSSG are shown after treatment with selenite for6-48 hours. Values represent the mean±SD for 3 experiments.

FIG. 7 shows the effects of selenite-induced growth inhibition andapoptosis in LAPC-4 prostate cancer cells and normal prostate cells.Panel A. LAPC-4 cells were treated with selenite at the indicatedconcentrations for 48 hours and cell proliferation was measured by MTSassay. Panel B. LACP-4 cells were treated with selenite for 48 hours andpercent apoptosis was detected as the fraction of cells in sub-G₁. PanelC, Caspase-3 cleavage in LAPC-4 cells following exposure to selenite for48 hours as detected by Western blot analysis. Actin protein expressionwas used to normalize for loading. Panel D, Primary cultures of normalprostate cells treated with selenite for 48 hours and percent apoptosiswas detected. Values represent the mean±SD for 3 experiments.

FIG. 8 shows results of analysis of MnSOD protein expression in primaryprostate (normal and cancer) cell strains. Western blot analysis ofMnSOD protein expression in E-PZ/CA-1, E-PZ/CA-2, and E-PZ/CA-3 cellsfollowing 48 hour exposure to selenite. Actin protein expression wasused to normalize for loading.

FIG. 9 shows the results of analysis of the effects of selenite on Bcl-2and Bax protein expression. Western blot analysis of Bcl-2 and Bax aexpression in Panel A LAPC-4 and Panel B E-PZ/CA-1, E-PZ/CA-2, andE-PZ/CA-3 cells following treatment with selenite for 48 hours. Actinprotein expression was used to normalize for loading.

FIG. 10 shows the effects of selenite on bcl-2, bcl-x_(L), and baxprotein expression. Western blot analysis of bcl-2, bcl-x_(L), and bax-αexpression in LAPC-4 cells after treatment with selenite for 48 hours.Actin protein expression was used to normalize for loading.

FIG. 11 shows the results of analysis of selenite sensitizes LAPC-4cells to irradiation. Clonogenic survival data for LAPC-4 cells treatedwith or without selenite (10 or 25 μM) for 6 hours prior to receiving 2Gy of irradiation. Colony-forming efficiency was determined 14 dayslater and surviving fractions were calculated. Values represent themean±SD for 3 individual experiments.

FIG. 12 shows results that illustrate that selenite enhancesradiation-induced cell killing in LAPC-4 cells. Clonogenic survival datafor LAPC-4 cells treated with radiation alone (closed squares) or 10 μMselenite for 6 hours (open squares) prior to 2 or 5 Gy of γ-irradiation.Surviving fractions were calculated as the plating efficiency of treatedcells divided by the plating efficiency of untreated cells. Forcombination experiments the results were normalized for the killing fromselenite alone. Values represent the mean±SD for 3 experiments.

FIG. 13 shows that selenite provides for radiosensitization of DU 145cells. Panel A, Cells were treated with 10 μM selenite for 6 or 12 hoursand changes in the ratio of GSH:GSSG were measured as describedpreviously. Panel B, Clonogenic survival data for DU 145 cells treatedwith radiation alone (closed squares) or 10 μM selenite for 6 (opensquares) or 12 (open triangles) hours prior to γ-irradiation. Survivingfractions were calculated as the plating efficiency of treated cellsdivided by the plating efficiency of untreated cells. For combinationexperiments the results were normalized for the killing from selenitealone. Values represent the mean±SD for 3 experiments.

DEFINITIONS

By “redox status (or state) of a cell” or “cellular redox status (orstate)” is meant the net effect of all oxidizing and reducing agents inthe cell, which is largely determined by the level of activity (e.g.,expression) of anti-oxidant enzymes in the cell. The redox status of acell is an important determinant of cellular responses to oxidativedamage, including reactive oxygen species (ROS). Most ROS, which canresult in oxidative damage originate from endogenous sources asby-products of normal and essential reactions, such as energy generationfrom the mitochondria.

“Reactive oxygen species” (ROS) are chemically reactive moleculesderived from oxygen and include superoxide (O₂.⁻), hydrogen peroxide(H₂O₂), and the hydroxyl radical (.OH). These highly reactiveintermediates can cause damage in numerous biological moleculesincluding proteins, lipids, and DNA. Excessive quantities of ROS canoverwhelm the buffering capacity of a cell, and create a pro-oxidantstate that predisposes the cell to undergo apoptosis. The alteration ofthe cellular redox state can also affect the activity of redox-sensitiveproteins via the oxidation of critical cysteine residues, which may inturn have downstream effects on signal transduction and genetranscription. The alteration of thiol status may be a key event duringapoptosis, and the distribution of thiols in intracellular compartmentsand in the mitochondrial membrane may modulate the cellular response tooxidative stress.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, and caninclude: (a) preventing the disease or a symptom of a disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it (e.g., including diseases that maybe associated with or caused by a primary disease); (b) inhibiting thedisease or condition, i.e., arresting its development (e.g., as ininhibiting tumor cell viability (e.g., cell division, growth, and thelike); and (c) relieving the disease, i.e., causing regression of thedisease (e.g., as in facilitating reduction in tumor size, tumor load,and the like), which can result in alleviation of symptoms, reduction ofthe severity of the disease (up to and including eradication of thedisease), cure of the disease, and the like.

The term “synergy” as used herein refers to a response to two or morestimuli that is greater than the sum of the response of the same stimuliapplied alone. For example, administration of an iSe compound and, forexample, a ROS-inducing cancer therapy (such as radiation) to tumorcells results in greater tumor cell killing (or growth inhibition) thancan be explained by the sum of the activity of either agent alone intumor cell killing (or tumor growth inhibition). Similarly, a“synergistic effect” is an effect that results from such a synergy.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, primates, including simians and humans.

The term “pharmacokinetic profile,” as used herein, refers to theprofile of the curve that results from plotting serum concentration of adrug over time, following administration of the drug to a subject.

Where a comparative term is used, such as “enhanced”, “increased”,“decreased”, and the like, such terms are used in reference to asuitable control condition. For example, where an iSe compound is saidto provide for “enhanced” sensitivity of a cancerous cell to anothercancer therapy, such “enhancement” is meant to refer to the sensitivityof the cancerous cells to the cancer therapy in the absence of iSecompound administration. In another example, where it is noted that acancerous cell has “elevated” GSH levels (e.g., as determined byGSH:GSSG ratio), such “elevation” is meant to refer to a GSH level in anon-cancerous cell, preferably of the same tissue type or origin. Theordinarily skilled artisan will readily appreciate the appropriatecomparison to be made when such references are made throughout thepresent specification.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acompound” includes a plurality of such compounds and reference to “thetumor” includes reference to one or more tumors and equivalents thereofknown to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that administration ofinorganic selenite provides for enhanced sensitivity of a tumor tocancer therapy, such as radiotherapy. Thus the invention featuresmethods and selenium-containing compositions for treating a neoplasticdisease in a subject. In particular, the invention features methods forenhancing sensitivity of a tumor to cancer therapy by treating the tumorwith an inorganic selenium-containing (iSe) compound and with a cancertherapy, particularly a cancer therapy that also affects the cellularredox status (e.g., generation of ROS) of a tumor cell (e.g.,radiation). Combination therapy with an iSe compound and another cancertherapy provides for a synergistic effect in tumor cell killing, whicheffect is tumor-specific (e.g., the combination does not act in asynergistic manner in killing non-cancerous cells).

The invention for the first time demonstrates that an inorganic seleniumcontaining compound (referred to herein as “iSe compound”), such asinorganic selenite, renders tumors more sensitive to other cancertherapies, such as radiotherapy, especially when administered prior toadministration of the second cancer therapy, e.g., so as to allow foriSe metabolism and alteration of the tumor cell redox state withaccompanying generation of ROS. Furthermore, the invention also providesa method that provides for a strong preference for killing cancerouscells. The invention finds particular use where the iSe compound isadministered in conjunction with, and particular prior to,administration of a cancer therapy that creates or provides reactiveoxygen species (ROS) in a cancerous cell.

In addition, the inventors have found that cancerous cells havingelevated levels of an antioxidant, such as glutathione (GSH), and/orelevated levels of Bcl-2 expression, are amenable to treatment using iSecompounds, either alone or in conjunction with administration of acancer therapy.

The inventors' findings have implications for clinical regimens Inshort, the present invention can provide for administration of lowerdoses, fewer doses, or both of cancer therapies, as the synergisticeffect in killing tumor cells (but not normal, non-cancerous cells) canbe exploited to both effect tumor killing while reducing toxicity andassociated side effects of cancer therapy.

More specifically, the inventors have found that inorganic seleniteinduces apoptosis in cancerous cells in a dose-dependent fashion.iSe-induced apoptosis is associated with decreased intracellularGSH:GSSG ratios (reduced glutathione (GSH) to glutathione disulfide(GSSG)). iSe-induced apoptosis in cancer cells appears to be associatedwith GSH depletion, but GSH depletion is not sufficient for iSe-inducedapoptosis. Superoxide production and altered redox signaling as a resultof iSe metabolism apparently contribute to iSe-induced apoptosis.

Furthermore, in contrast to apoptosis-inducing agents such as BrefeldinA, normal (non-cancerous) cells are resistant to iSe-induced apoptosis(relative to cancerous cells), while cancer cells were sensitive toiSe-induced apoptosis. Agents such as Brefeldin A do not discriminatebetween normal and cancerous cells in induction of apoptosis. Moreover,primary cultures of prostate cancer cells were found to have asignificantly higher (>3-fold) intracellular GSH:GSSG ratio than thosecells derived from matched normal tissue. Thus, the inventors have founda direct relationship between the level of GSH in the cell and thepotency of selenite in exerting cytotoxic effects. The rank order ofsensitivity to selenite in cells tested coincided with intracellular GSHstatus.

The inventors have also shown that selenite inhibits Bcl-2 expressionand induces Bax-α (mitochondrial targeted Bax) expression in cancercells, while normal cells, which are more resistant to the effects ofiSe, show increased Bcl-2 protein and no change in Bax-α expression inresponse to iSe. Without being held to theory, levels of manganesesuperoxidedismutase (MnSOD) may have a protective effect in normalcells, since the inventors observed that, in the context of prostatecancer, normal prostate cells had a significantly higher level of MnSODexpression than prostate cancer cells. Furthermore, and again withoutbeing held to theory, the relatively lower level of MnSOD in cancerouscells compared to non-cancerous cells can contribute to the toxiceffects of iSe on prostate cancer cells.

Patients having cancerous cells with elevated Bcl-2:Bax ratios (relativeto such ratios in non-cancerous cells) are at increased risk ofradiation therapy failure. Since the inventors have found that cancerouscells treated with selenite prior to receiving ionizing radiation (IR)showed significantly higher levels of apoptosis compared to cellstreated with selenite or IR alone, patients having tumors with elevatedBcl-2:Bax ratios can be particularly amenable to treatment according tothe invention.

Thus, the invention also provides a means to assess a patient's tumor,and select a course of therapy that is most likely to succeed. AssessingGSH levels, MnSOD levels, and Bcl-2 levels in cancerous cells of thepatient (relative to normal cells of the patient), allows the clinicianto select a therapy that is best suited for the patient. The inventionin this aspect allows the clinician to avoid administration of therapiesdoomed to failure. In addition, the methods of treatment according tothe invention can provide for a shortened course of treatment thatprovides for a successful result.

The iSe combination therapy of the invention provides the unexpectedadvantage that iSe compounds provide for sensitization of tumor cells tocancer therapy (e.g., radiation therapy, chemotherapy, particularly aROS-inducing cancer therapy)—but not normal cells to a significantdegree. In addition, the anti-tumor, tumor-specific effects of iSecompounds are maintained in the presence of the second cancer therapy.Furthermore, and unexpectedly, the effects of combination therapy withan iSe compound and another cancer therapy are synergistic. Thesephenomenon can operate together to provide for, for example, lower orfewer doses of iSe required to inhibit tumor cell proliferation, tumorgrowth, or tumor cell survival; lower or fewer doses of the selectedsecond cancer therapy required to inhibit tumor cell proliferation,tumor growth, or tumor cell survival; and lower or fewer doses of boththe iSe compound and the second cancer therapy, where the doses arelower or fewer than doses required in a therapy involving administrationof an iSe compound or the second cancer therapy alone.

The invention will now be described in more detail.

Inorganic Selenium-Containing Compounds and Formulations

Inorganic selenium-containing compound suitable for use in the inventioncan be provided in a variety of forms. It is noted that selenium may bepresent in elemental form or as inorganic or organic selenium compounds.It is also noted that selenium occurs in a number varying valence forms.For example, selenium compounds occur in which the selenium has a +4valance or a +6 valence, as the selenite and selenate ions,respectively. Preferably, the selenium-containing compound is aninorganic selenium-containing compound, referred to herein as an “iSecompound”. It is to be understood, however, that the particularinorganic forms of selenium compounds set forth herein are not to beconsidered limitative.

Among the inorganic selenite and selenate forms, of interest for use inthe compositions of this invention are the water soluble alkali metalsalts thereof, and particularly, the sodium and potassium salts, thatis, sodium and potassium selenite and selenate. Of particular interestfor use in the compositions of this invention are the water solublealkali metal salts of selenite, and particularly, the sodium andpotassium salts, that is, sodium selenite and potassium selenite.

As noted above, selenium compounds may be present in organic forms,which can be referred to as “organoselenium compounds”. Exemplaryorganoselenium compounds include selenium compounds of cysteine andmethionine, as well as an organic selenium compound selected from thegroup consisting of RSeH, RSeR, RSeR′, RSeSeR and RSeSeR′, wherein R andR′ are the same or different and each is an aliphatic residue containingat least one reactive group selected from the group consisting ofaldehyde, amino, alcoholic, carboxylic, phosphate, sulfate, halogen orphenolic reactive groups and combinations thereof. Use of organoseleniumcompounds is less preferred, and may be explicitly excluded from use inthe compositions of the invention. Thus, formulations comprising iSecompounds for use in the invention may lack organoselenium compounds ina therapeutically effective amount (e.g., an amount insufficient toeffect significant cancerous cell growth inhibition or killing) or lacka detectable organoselenium compounds, such as selenomethionine orselenocystine, and derivatives thereof

The iSe compounds for use in the invention are generally water solubleinorganic selenite or selenate compounds, such as alkali metal salts ofselenite or selanate. Of particular interest are water soluble inorganicalkali metal selenites, and particularly, the sodium and potassiumsalts, that is, sodium selenite and potassium selenite. Use of sodiumselenite is of particular interest.

iSe compounds are generally administered to a subject in an amounteffective to inhibit cancerous cell growth (including cancerous cellkilling), preferably with insignificant or relatively little inhibitionof normal cell growth (including normal cell killing). In someembodiments, iSe is administered in an amount effective to enhancesensitivity of the cancerous cells to another cancer therapyadministered in conjunction with administration of iSe compound.

In exemplary embodiments, iSe can be administered in an amount of, forexample, about 0.25 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 1.5mg/kg, about 3.0 mg/kg, about 6.0 mg/kg, or more with the proviso that adesirable therapeutic index is achieved while providing for alterationof the redox state in the cancerous cell. In some embodiments, iSecompound is administered in amounts ranging from 0.5 mg/kg to 4.0 mg/kg,usually 1.0 mg/kg to 3.0 mg/kg. In some embodiments, the amount of iSecompound administered is greater than 1.7 mg/kg, or greater than 2.0mg/kg. Doses of iSe are generally greater than that normally associatedwith use of iSe as a chemopreventive agent, and can be greater than thatused in supportive cancer therapy (in which iSe is administered as aprotective agent for normal cells in cancer therapy, but not as ananti-cancer agent itself). For example, iSe compounds can beadministered at doses greater than 200 μg per day (e.g., for a 75 kbindividual, usually by oral administration). Doses of iSe compounds forthe therapies of the invention are generally about 10 to 20 fold greaterthan doses conventionally administered for chemoprevention.

Doses of iSe in accordance with the invention can be administered inwhole or divided doses, and can may be administered daily, thriceweekly, twice weekly, weekly, and the like, with daily or weekly dosingbeing of particular interest. Where iSe compounds are administered inconjunction with another cancer therapy, the iSe dose can beadministered at the time of or prior to administration of the cancertherapy and in a dosing schedule that corresponds to the dosing regimenof the other cancer therapy, e.g., so that iSe compound is administeredat the time of or prior to each dose of the other cancer therapy.

It will be appreciated that amounts of iSe administered will vary with avariety of factors including, but not limited to, whether the iSecompound is used as a monotherapy (e.g., alone, and not necessarily withother cancer therapies) or in conjunction with another cancer therapy(e.g., radiation, chemotherapy, and the like), form of iSe administered(e.g., selinite or selenate, salt of iSe, and the like), route ofadministration, formulation, dosage form, severity or extent of disease,tumor type (e.g., localized, metastatic, tissue or origin, and thelike), and other factors that will be readily appreciated by a clinicianor other health care practitioner.

The therapeutic compositions in accordance with the present inventionmay be provided in various physical forms, for a variety of methods androutes of administration. For example, the composition can be formulatedfor, for example, injectable (parenteral), oral, topical, mucosal, andsuppository administration. Also of interest are administration byintravenous, intratumoral, tumor targeted, or peritumoral routes, aswell as administration to the vasculature of a tumor bed.

The iSe compound-containing compositions of the invention may compriseinert or active additives. For example, the compositions of the canfurther comprise a suitable pharmaceutically acceptable excipient, whichmay be a vehicle, carrier, diluent, and/or adjuvant. The compositionscan further comprise pharmaceutically acceptable auxiliary substances,such as pH adjusting and buffering agents, tonicity adjusting agents,stabilizers, wetting agents and like, are readily available to thepublic. The selection of such suitable additional components will dependupon, for example, the form desired, the route of administration, andthe neoplastic disease to be treated. The additional components aregenerally selected so as to not detrimentally affect any of the activeingredients of the composition.

Exemplary inert carriers or vehicles include: sugars and milk sugars,such as lactose; liquids, such as water, isotonic aqueous solutions,saline solutions and alcohol; and inert powders, creams, salves,ointments, cleansing and antiseptic agents and the like.

Exemplary pharmaceutically active additional components may includeother cytotoxic agents, e.g., chemotherapeutic drugs, biologicalresponse modifiers, or radiosensitizers. As used herein, the term“biological response modifier” (BRMs) refers to compounds which are, intheir naturally-occurring state, produced in small amounts as part ofthe body's natural response to cancer or other diseases. Exemplary BRMsinclude monoclonal antibodies that bind to antigen of a malignant cells,and which may have an attached cytotoxic molecule (e.g., toxin,radioactive component, etc.); and cytokines (e.g. interferons,interleukins, colony-stimulating factors CSFs) which can stimulate bloodcell production and help restore blood cell counts more rapidly aftertreatment. BRMs can be isolated, naturally-occurring molecules orrecombinantly or otherwise artificially produced. Examples of thesedrugs include, but not limited to Rituxan, HER-2, CMA-676, IFN-α (e.g.,IFN-α2a, IFN-α2b, consensus interferon) Interleukin-2, Interleukin-3,Erythropoetin, Epoetin, G-CSF, GM-CSF, Filgrastim, Sargramostim andThrombopoietin, as well as modified forms (e.g., PEGylated andhyperglycosylated forms) of such molecules. See, e.g., U.S. PatentPublication No. 20020107225, incorporated by reference herein.

In the subject methods, the iSe compounds may be administered to thehost using any convenient means capable of resulting in the desiredtherapeutic effect. Thus, the agents can be incorporated into a varietyof formulations for therapeutic administration. More particularly, theiSe compounds, in combination with appropriate, pharmaceuticallyacceptable excipients (e.g., carriers or diluents), may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants and aerosols. In general, iSecompounds for use in the invention are formulated for enteraladministration (e.g., by oral, oral, buccal, or rectal administration),or parenteral administration (e.g., by subcutaneous, intradermal,intraperitoneal, intravenous, or intramuscular administration, e.g.,infusion or injection). Administration may also be accomplished by, forexample, transdermal, intratracheal, or inhalation administration.

In pharmaceutical dosage forms, the iSe compounds may be administered inthe form of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

The iSe compounds can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

In the case of the injectable form of iSe compounds, the compounds maybe dissolved in an aqueous buffer to form a parenteral preparation.Suitable aqueous buffers include, but are not limited to, acetate,succinate, citrate, phosphate buffers varying in strength from 5 mM to100 mM, and distilled or sterilized water. In some embodiments, theaqueous buffer may include sodium chloride, and sugars e.g., mannitol,dextrose, sucrose, glucose and the like.

For oral preparations, the iSe compounds can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

Furthermore, the iSe compounds can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature. iSe compounds can also be providedin sustained release or controlled release formulations, e.g., toprovide for release of agent over time and in a desired amount (e.g., inan amount effective to provide for a desired therapeutic or otherwisebeneficial effect).

Unit dosage forms for oral or rectal administration also include, forexample, syrups, elixirs, and suspensions may be provided wherein eachdosage unit, for example, teaspoonful, tablespoonful, tablet orsuppository, contains a predetermined amount of the compositioncontaining one or more inhibitors. Similarly, unit dosage forms forinjection or intravenous administration may comprise the inhibitor(s) ina composition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

It is to be understood that the particular carriers or vehicles set outabove are illustrative only and other known pharmaceutically acceptablematerials can be utilized in the compositions of this invention so longas they do not adversely react or interact with the iSe compounds andother active ingredients to destroy the identity or activity thereof.Moreover, the particular carrier or vehicle chosen for use will dependupon the form of the composition needed for the particular method ofadministration and the host to receive the composition.

In those cases where the composition contains a larger amount of the iSecompound (e.g., in some embodiments more than about 1.0 mg by weight),the composition may be employed in the form of divided dosages whenbeing administered whether it be in the form of a tablet, a capsule or aliquid solution. Moreover, a particular dosage in this respect can beadministered several times a day so long as the total amount of iSecompound does not exceed a generally accepted maximum dosage.

In some instances, the composition of this invention can be made bysimply mixing the selenium compound in proper proportion with anappropriate carrier. For example, in preparing tablets, an alkali metalselenite or selenate salt in its dry form may be mechanically mixed witha powdered carrier or vehicle and shaped or pressed into tablets orencapsulated by known art recognized techniques. On the other hand, ifdesirable, such salts can be dissolved in water and then mixed with apowdered carrier and shaped or pressed into tablets.

As an alternative, liquid compositions can be prepared simply bydissolving the iSe compound in water and using the composition in thatform with recognized additives for either external or oral application.The materials as mixed should contain a desired amount of iSe, which ina single or divided dose achieve a desired therapeutic effect.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of the agentscalculated in an amount sufficient to produce the desired effect inassociation with a pharmaceutically acceptable diluent, carrier orvehicle. The specifications for the unit dosage forms for use in thepresent invention depend on the particular compound employed and theeffect to be achieved, the pharmacodynamics associated with eachcompound in the host, and the like.

Administration of iSe Compounds as a Monotherapy or in Conjunction withOther Cancer Therapies

In one embodiment, the invention provides a method of treating aneoplastic disease in a subject by administering an iSe compound inconjunction with a second cancer therapy, e.g., a cancerchemotherapeutic drug, cancer radiotherapy, and the like. Thisembodiment may be referred to herein as “iSe combination therapy”. Thesecond cancer therapy is preferably one that alters the redox(reduction-oxidation) state of the cell toward oxidation, e.g., throughgeneration of superoxide or other oxidative stress, e.g., by productionof reactive oxygen species (ROS), oxidations of antioxidants in the cell(e.g., thiol-containing antioxidants, such as glutathione), and thelike.

Exemplary Cancer Therapies for Use in the Invention

Cancer therapies for use in iSe compound combination therapy asdescribed herein can be selected from any available cancer therapy, andselected according to factors such as the cancer type to be treated. Ofparticular interest is administration of a cancer therapy that createsor provides reactive oxygen species (ROS) in a cancerous cell,particular those that depletes GSH in a cancerous cell. Such cancertherapies are referred to herein as a “ROS-inducing cancer therapy”.Preferably, the iSe is administered prior to administration of thecancer therapy. Treatment regimen in the context of iSe combinationtherapy are discussed in more detail below.

Cancer therapies that can be used in combination with iSe compoundtherapy include, but are not limited to, DNA damaging agents such as DNAintercalating agents, DNA alkylating agents, and DNA modifying agents,nucleic acid biosynthesis inhibiting agents, apoptosis inducing agents,and mitosis inhibiting agents. Exemplary cancer therapies include, butare not limited to, cyclophosphamide, estramustine, paclitaxel,vinblastine, and cisplatin.

As noted above, administration of a ROS-inducing cancer therapies inconjunction with administration of iSe compounds is of particularinterest. Exemplary ROS-inducing cancer therapies include, but are notnecessarily limited to, radiation therapy, chemotherapy, photodynamictherapy (also called PDT, photoradiation therapy, phototherapy, orphotochemotherapy), administration of ROS-inducing biological responsemodifiers, and the like.

Where the cancer therapy to be administered is a ROS-inducingchemotherapy, suitable exemplary chemotherapeutic agents include, butare not limited to, inhibitors of glutathione synthesis, inhibitors ofother antioxidant enzymes, and inhibitors of the anti-apoptotic Bcl-2family members, Akt or other important regulators of apoptosis.

Exemplary commercially available compounds include, but are notnecessarily limited to: buthionine sulfoximine; anthracyclines,adriamycin (doxorubicin), adriamycinone (doxorubicinone), daunomycin,daunomycinone, daunorubicin, and daunorubicin derivatives such as5-iminodaunorubicin, ubiquinone, Acid Blues 25, 80, and 41, Acid Green25, anthraquinone and its derivatives such as 2-bromoanthraquine,1,2-dihydroxyanthraquinone, 1,8-diaminoanthraquinone,2,6-diaminoanthraquinone, 1,5-dichloroanthraquinone,1,2-diaminoanthraquinone, and 2-chloro-anthraquinone, quinizarin,anthrarufin, quilalizarin, aloe-emodin and related compounds such as5-nitro-aloe-emodin, 5-amino-aloe-emodin. 2-allylaloe-emodin, averufin,kalafungin, alizarin complexone dihydrate, quercetin dihydrate, acidblack 48, procytoxid, leucotrofina, azimexon, andmethoxycin-narnonitrile.

Where the cancer therapy to be administered in conjunction with iSecompound therapy is radiation, radiation therapy can be accomplishedaccording to conventional methods. Such methods include externalradiation therapy, brachytherapy (internal radiation therapy), andtargeted radiation.

Administration of Cancer Therapy in Connection with iSe CompoundAdministration

In accordance with the iSe combination therapy of the invention, thesecond cancer therapy can be administered in conjunction with the iSecompound in a variety of ways. “In conjunction”, is meant to encompassadministration of iSe prior to or at the time of (preferably, usuallynot subsequent to) administration of the second cancer therapy.Preferably iSe is administered prior to or at the time of administrationof the second cancer therapy, more preferably, iSe is administered priorto administration of the second cancer therapy.

In a particularly preferred embodiment, the iSe is administered prior tothe second cancer therapy, and in an amount sufficient to provide foriSe-induced alteration of the tumor cell redox state toward oxidation.The second cancer therapy is then administered after iSe has inducedalteration of the tumor cell redox state, e.g., after a time sufficientfor metabolism of iSe. The redox state of the tumor cell can bedetermined by, for example, a reduction in GSH levels in a tumor cell(e.g., a reduction in GSH:GSSH ratio), a decrease in Bcl-2 expressionlevels (e.g., as detected by a decrease in Bcl-2:Bax expression levels),and the like. For example, second cancer therapy is optimallyadministered when the ratio of GSH:GSSG in a cancerous cell is reducedleast about 25%, 30%, 40%, 50%, 60%, 75%, 85%, 90% or more, usually atleast about 50% (relative to prior to iSe compound administration).

The iSe combination therapy of the invention provides the unexpectedadvantage that iSe compounds provide for sensitization of tumor cells tocancer therapy (e.g., radiation therapy, chemotherapy) but not normalcells to a significant degree, particularly where iSe compound isadministered prior to administration of the second cancer therapy. Inaddition, the anti-tumor, tumor-specific effects of iSe compounds aremaintained in the presence of the second cancer therapy. Furthermore,and unexpectedly, the effects of combination therapy with an iSecompound and another cancer therapy are synergistic. These phenomenoncan operate together to provide for, for example, lower or fewer dosesof iSe required to inhibit tumor cell proliferation, tumor growth, ortumor cell survival; lower or fewer doses of the selected second cancertherapy required to inhibit tumor cell proliferation, tumor growth, ortumor cell survival; and lower or fewer doses of both the iSe compoundand the second cancer therapy, where the doses are lower or fewer thandoses required in a therapy involving administration of an iSe compoundor the second cancer therapy alone.

Neoplastic Disease Amenable to Therapy

Neoplastic disease contemplated for treatment according to the methodsof the invention include any abnormal growth of tissue, which may bebenign or cancerous, with treatment of cancerous neoplastic diseasebeing of most interest. Reference to “cancer” or “tumor” herein is notmeant to be limiting, but only exemplary of the disease to which thetherapy of the invention can be applied. Tumors susceptible to therapyaccording to the invention are generally any tumor that, for example,once sensitized with an iSe compound according to the invention, can betreated using another cancer therapy, particularly so as to provide asynergistic or enhanced effect of the combination of the iSe and cancertherapy.

As discussed above, as well as in the Examples below, normal(non-cancerous) cells are relatively resistant to iSe-induced apoptosis,while cancer cells were sensitive to iSe-induced apoptosis. Furthermore,the inventors have found a direct relationship between the level of GSHin the cell and the potency of selenite in exerting cytotoxic effects.The rank order of sensitivity to selenite in cells tested coincided withintracellular GSH status.

The inventors have also shown that selenite inhibits Bcl-2 expressionand induces Bax-α (mitochondrial targeted Bax) expression in cancercells, while normal cells, which are more resistant to the effects ofiSe, show increased Bcl-2 protein and no change in Bax-α expression inresponse to iSe. Without being held to theory, levels of manganesesuperoxidedismutase (MnSOD) may have a protective effect in normalcells, since the inventors observed that, in the context of prostatecancer, normal prostate cells had a significantly higher level of MnSODexpression than prostate cancer cells.

Thus, susceptibility of a tumor to treatment using iSe-based therapy andcombination therapy can be assessed by assessing the GSH:GSSG ratio orthe Bcl-2:Bax expression level ratio in the candidate tumor cells. Tumorcells having elevated GSH levels relative to a normal GSH level (e.g.,compared to a matched normal cell control), or having elevated Bcl-2expression levels relative to a normal Bcl-2 expression level (e.g.,compared to a matched normal cell control), are susceptible to iSe-basedtherapy according to the invention. Methods for assessing GSH levels andfor assessing Bcl-2 expression levels are well known in the art, withexemplary assays described in the Examples section below.

Thus, in another embodiment, the invention features, methods foridentification of a patient having a tumor susceptible to the iSecombination therapy of the invention. Such methods involve assessing alevel of GSH in a cancerous cell of the patient, and comparing the levelof GSH in the cancerous cell with a normal level of GSH (e.g., in anormal cell, e.g., a matched normal cell from the same tissue type andthe same patient). If the GSH level is greater in the cancerous cellthan a normal GSH level, then the patient is amenable to therapy usingan iSe combination therapy of the invention. In a related embodiment,GSH levels are assessed by examining the GSH:GSSG ratio (reducedglutathione to glutathione disulfide). Methods for assaying GSH and GSSGare well known in the art.

In a related embodiment, suitable patients for therapy according to theinvention are identified by assessing a level of Bcl-2 expression in acancerous cell of the patient, and comparing the level of Bcl-2expression in the cancerous cell with a normal level of Bcl-2 expression(e.g., in a normal cell, e.g., a matched normal cell from the sametissue type and the same patient). If the Bcl-2 level is greater in thecancerous cells than in normal cells, then the patient is amenable totherapy using an iSe combination therapy of the invention. Bcl-2 and Baxexpression levels can be assessed and the Bcl-2:Bax ratio calculated.Methods for assaying Bcl-2 and Bax expression levels are well known inthe art.

In a further related embodiment, the invention provides foridentification of a patient suitable for iSe therapy by assessingantioxidant levels in normal cells and in cancerous cells. As notedabove, if levels of an antioxidant, such as MnSOD, are higher in normalcells than cancerous cells, the patient is amenable to iSe therapy, asthe antioxidant should serve to protect normal patient cells from theeffects of the therapy, while at the same time effecting killing ofcancerous cells.

In general, the tumors can be solid tumors or lymphohematopoietictumors. Tumors can be of any tissue origin, grade or stage. For example,the tumors can be associated with cancer of the breast, colon,endometrium, head and neck, lung, skin (e.g., melanoma, basal cellcarcinoma, squamous cell carcinoma, and the like), digestive system,gastrointestinal system (including colon, rectum, etc), oral cavity(including lip, mouth, etc.), musculoskeletal system (including muscle,bone, etc.), endocrine system, eye, genitourinary system (e.g., bladder,prostate, ovary, etc.), neurologic system (e.g., brain, etc.), and thelike. Both hormone-responsive and hormone-resistant cancers (e.g.,androgen-responsive and androgen-resistant prostate cancer;estrogen-responsive and estrogen-resistant breast cancer) and p53 wildtype and mutant cancers are of interest for treatment. Treatment ofprostate cancer and ovarian cancer, especially cancer therapy resistantprostate and ovarian cancers, are of particular interest, both in thecontext of administration of iSe compounds alone and in combinationtherapy (e.g., in conjunction with administration of a second cancertherapy) according to the invention. Such treatment of prostate canceris of especial interest.

The methods of the invention can be used to treat primary tumors ormetastases. In addition, the tumors may be either malignant or benign,with radiation therapy generally being used to treat malignant tumors.The invention finds particular use in the treatment of tumors that aregenerally recognized as being resistant to therapy or which have provenin a particular patient to be resistant to cancer therapy, e.g., asevidenced by relapse, recurrence, or non-responsiveness to prior cancertherapy. Where the tumor has proved or is known to be resistant to achemotherapeutic drug, such tumors are often referred to as“drug-resistant” tumors.

Of particular interest are those cancers that are recognized to be orproved resistant to, or relapsed after, administration of a conventionalcancer therapy (e.g., radiotherapy, chemotherapy, etc.) that did notinclude iSe compound combination treatment, particularly where the priortherapy did not involve treatment with an iSe compound followed byadministration of a different cancer therapy, according to theinvention. Accordingly, treatment of patients who have failed priortherapy (“treatment failure” patients), which patients either initiallyresponded then relapsed (e.g., did not have sustained remission) or whodid not significantly respond (e.g., had tumors that proved resistant toconventional therapy), are of particular interest for treatmentaccording to the methods of the present invention. In one embodiment,the goal of invention is to 1) sensitize tumors to a subsequent cancertherapy by administration of an iSe compound; and/or 2) provide for asynergistic effect on tumor growth inhibition by a combination therapyof an iSe compound administered either prior to or with another cancertherapy, particularly a cancer therapy that provides for alteration oftumor cell redox state toward oxidation (e.g., through depletion of GSHlevels (e.g., reduction of GSH:GSSG ratio), reduction of Bcl-2expression levels (e.g., reduction of Bcl-2:Bax expression level ratio),and the like).

Dosing Regimen for iSe Combination Therapy

In general, an “effective amount” of an iSe compound, and particularlywhere administered alone (e.g., not necessarily in conjunction with asecond cancer therapy) is an amount facilitates growth inhibition, up toand including killing, of tumor cells in a subject. Furthermore, in thecontext of iSe combination therapy, an “effective amount” of an iSecompound is generally an amount that can significantly increase thesensitivity of a tumor to subsequent or concomitant cancer therapycompared to similarly treated tumors without iSe compound treatment(e.g., compared to an expected sensitivity of the tumor type in theabsence of iSe compound administration) (P value<0.05).

Where iSe compounds are to be administered prior to administration ofanother cancer therapy, iSe compounds can be administered according tothe invention as early as 2 hours, 6 hours, 12 hours, 24 hours, 2 days,prior to initiation of the second cancer therapy. The second cancertherapy can be administered either as fractionated or single dosetherapy, preferably fractionated therapy. Normally iSe compound isadministered within about 12 hours, 24 hours, 48 hours, 36 hours, 72hours prior to the second cancer therapy, usually about 48 hours priorto the second cancer therapy, and can be administered on the same daywhen the second cancer therapy is initiated.

Precise dosage regimens will vary according to a variety of factors asdiscussed above, including in this context the course of the second(e.g., subsequent or concomitant) cancer therapy prescribed, and, theiSe compound formulation to be administered.

Where the cancer therapy to be administered in conjunction with iSecompound therapy is radiation, radiation therapy can be accomplishedaccording to conventional methods. Radiation can be administered in asingle dose or more commonly in fractionated doses, with the latterbeing of particular interest. For example, fractionated radiationtherapy is generally administered daily, 5 days per week forapproximately 2 weeks, 4 weeks, 5, weeks, 7 weeks, 8 weeks, 9 weeks, ormore usually for approximately about 2 to 7.5 weeks, with the specificdosing regimen dependent upon a number of factors including thehistological type of tumor and stage of disease.

Where the cancer therapy to be administered in conjunction with iSecompound therapy is a chemotherapeutic drug, administration can beaccomplished according to conventional methods. The chemotherapeuticdrug can be administered in a single dose or in fractionated doses. Forexample, chemotherapy is generally administered once about every 2weeks, 3 weeks, or 4 weeks, for a total 4 courses, 5, courses, 6courses, 7 courses, or 8 courses, with the specific dosing regimendependent upon a number of factors including the histological type oftumor and stage of disease.

In one embodiment of the invention, administration of iSe compoundsenhances tumor sensitivity to cancer therapy such as radiation therapyor chemotherapy, and thus increases the efficacy of conventional cancertherapies. Administration of iSe compounds also provides for enhancementof cancer therapy in a manner that is more specific to tumor cells,i.e., administration of iSe compounds does not significantly increasesensitivity of non-cancerous cells to cancer therapy, e.g., radiation orchemotherapy.

The invention can thus, in some embodiments, allow for shortened coursesof cancer therapy, reduction in doses of required for therapy (e.g.,reduction in doses of iSe compounds, radiation, chemotherapeutic drugs)required to achieve a given outcome (e.g., compared to a dose requiredin the absence of iSe compound administration or in the absence ofcombination therapy with iSe). For example, administration of an iSecompound in conjunction with radiation therapy or chemotherapy may allowthe clinician to administer reduced doses of radiation (e.g., about 75%,80%, or 90-95% of a conventional dose in the absence of iSe combinationtherapy), with doses that can be increased or decreased as needed (e.g.,relative to the apparent sensitivity of the tumor). Alternatively or inaddition, administration of an iSe compound in conjunction withradiation therapy or chemotherapy can allow for reduced doses of iSecompounds (e.g., about 75%, 80%, or 90-95% of a conventional dose in theabsence of radiation combination therapy). Alternatively or in addition,administration of an iSe compound in conjunction with radiation therapycan allow for shortening of the course of therapy (e.g., by reducing thetotal number of treatments required to eradicate a tumor by 1, 2, 3, 4,5, or more)).

In some embodiments, therapy involves administration of an amount of iSecompound and dose of a cancer therapy effective to eradicate the tumorwhen treating with curative intent or achieve local control/palliatesymptoms when treating with palliative intent. For example, therapyaccording to the invention can involve killing all of the tumor cells inthe radiation field, shrinking the tumor by killing some of the cells,or controlling tumor growth (e.g., so as to maintain the tumor at itssize at the initiation of therapy), by decreasing or delaying tumorgrowth.

The effectiveness of iSe compound in potentiating radiation therapy orchemotherapy, or otherwise providing for a synergistic effect due to thecombination therapy, can be assessed by conventional methods. Forexample, tumor size (e.g., tumor volume) can be assessed by physicalexamination in some cases and by the use of imaging techniques such asMRI, CT, ultrasound, PET and the like.

iSe compound combination therapy (i.e., iSe compounds administered inconjunction with radiation therapy, chemotherapy, or both) can becombined with other treatment regimens as desired, and may beparticularly useful in subjects receiving combined modality therapy(either sequential or concurrent chemo and radiation therapy, with orwithout biological response modifiers). For example, iSe compounds canbe administered in conjunction with both a chemotherapy treatment andwith radiation therapy, either as separate or combined therapies. Forexample, a patient may receive an iSe compound, then radiation therapy;an iSe compound, then chemotherapy; an iSe compound, then chemotherapy,and then radiation therapy; an iSe compound, then radiation therapy,then chemotherapy, or an iSe compound together with both chemotherapyand radiation therapy.

Kits

Kits with unit doses of a subject iSe compound-containing formulationssuitable for use in the invention, e.g., in injectable dose(s), areprovided. In such kits, in addition to the containers containing theunit doses will be an informational package insert describing the useand attendant benefits of the iSe compound formulations in treatingneoplastic disease when administered in conjunction with a second,suitable cancer therapy. The kit can include, for example, dosingregimen for the iSe compound formulation and for a variety of second,suitable cancer therapies, where the dosing regimen is one that providesfor the most enhanced or synergistic effect of the combination therapy.

In some embodiments, a subject kit includes a container comprising asolution comprising a unit dose of an iSe compound formulation,particularly sodium selenite, and a pharmaceutically acceptableexcipient; and instructions to administer a unit dose according to adesired regiment or exemplary regiment dependent upon tumor type, age,weight, second cancer therapy (e.g., radiotherapy, chemotherapy, etc.)and the like. Providing iSe compound formulation suitable forintravenous administration is of particular interest, and in suchembodiments the kit may further include a syringe or other device toaccomplish such administration, which syringe or device may bepre-filled with the iSe compound formulation. The instructions can beprinted on a label affixed to the container, or can be a package insertthat accompanies the container.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric.

Examples 1-4 Effects of Selenite on Prostate Cancer Cells

The following examples present analysis of the effects of selenite onLAPC-4 prostate cancer cells, DU-145 prostate cancer cells, and matchedpairs of primary prostate (normal and cancer) cell strains in vitro.Prostate cancer cells were found to be significantly more sensitive toselenite-induced apoptosis than normal prostate epithelial cells.Without being held to theory, the data presented below from mechanisticstudies suggest that the differential response of normal and cancercells is due, at least in part, to differences in the relativeexpression of antioxidants (GSH and MnSOD) and Bcl-2 and DU-145 familymembers. Furthermore, the data below show that selenite can sensitizeLAPC-4 cells to γ-irradiation. In addition, synergistic effects ofselenite and radiation in delay of tumor growth were observed in anon-human animal model.

Material and Methods

The following materials and methods are used in the Examples below.

Cell Cultures and Treatments.

LAPC-4 prostate cancer cells (provided by Dr. Charles Sawyers, UCLA)were cultured in phenol red free RPMI 1640 (Life Technologies,Rockville, Md.) supplemented with 10% fetal bovine serum (GeminiBio-Products, Woodland, Calif.) at 37° C. in a humidified atmospherewith 5% CO₂.

Primary prostate epithelial cell strains from normal prostate (PZ) andprostate cancer (CA) tissue were provided by Dr. Donna Peehl (StanfordUniversity) using well established methods she has previously described(Peehl, Human prostatic epithelial cells. In: Culture of EpithelialCells, 2^(nd) edition, Freshney, R. I. and Freshney, M. G. (eds.),Wiley-Liss, Inc., N.Y., 171-194 (2002)). Three matched pairs of normaland cancer cell strains were available for study (E-PZ/CA-1, E-PZ/CA-2,and E-PZ/CA-3). Normal cell strains were obtained from a region of theprostate peripheral zone not involved in cancer. E-CA-1, E-CA-2, andE-CA-3 cell strains were derived from prostate carcinomas. Cells weretreated at 50-70% confluence.

DU 145 prostate cancer cells (American Type Culture Collection,Rockville, Md.) were cultured in RPMI 1640 (+phenol red) supplementedwith 10% fetal bovine serum. When the cells reached 50% confluence themedia was replaced and selenite was added at the noted concentrations.

Sodium selenite (referred to in the Examples below as “selenite” forconvenience) and buthionine sulfoximine (BSO) were obtained from Sigma(St. Louis, Mo.) and prepared in dH₂0. A ¹³⁷Cs irradiator was used todeliver γ-irradiation. All chemicals and reagents were supplied by Sigma(St. Louis, Mo.) unless otherwise noted.

Cell proliferation assay. Cells were seeded in 96-well plates (10,000cells/well) and treated with selenite for 48 hours. Cell survival wasassayed using the CellTiter 96 AQ_(ueous) One Solution CellProliferation Assay (Promega, Madison, Wis.). The MTS tetrazoliumsolution was added directly to the wells, incubated at 37° C. for 3hours, and then the absorbance was read at 490 nm with a 96 well platereader (Vmax Kinetic Microplate Reader, Molecular Devices, Sunnyvale,Calif.).

Detection of Apoptosis.

Flow cytometry analysis (FACS) analysis of propidium iodide (PI)-stainedcells was used to quantify the percentage of apoptotic cells as thefraction of cells with a hypodiploid amount of DNA (Sub-G₁). Cells werefixed and permeabilized with 100% ice-cold ethanol at 4° C. overnight.The cells were resuspended in 500 μl of buffer (phosphate bufferedsaline (PBS), 5 mM EDTA) and incubated with 200 μg/ml RNase A for 30 minat room temperature and then 50 μg/ml PI for 10 min at room temperature.The cell cycle distribution was analyzed using Facstar flow cytometry(Becton-Dickinson, San Jose, Calif.). Cleavage of Caspase-3 as a markerof caspase-mediated apoptosis was also detected by immunoblot analysis(see below).

Western Blot Analysis.

Cells pellets (containing floating and adherent cells) were resuspendedin lysis buffer (50 mM HEPES pH 7.5, 0.5% NP-40, 0.5% sodiumdeoxycholate, 50 mM sodium chloride, 1 mM EDTA and 0.1 mM sodiumorthovanadate), incubated on ice for 20 min, and spun at 14,000×g tocollect whole cell lysates. Protein content was determined using the Dcprotein assay (Bio-Rad, Richmond, Calif.). Total cell lysates (15 μg)was run on NuPAGE 10% Bis-Tris gels (Invitrogen, Carlsbad, Calif.).Proteins were transferred to PVDF membranes and blocked with 5%milk/Tris buffered saline (100 mM Tris-HCl pH 7.5, 150 mM NaCl/0.1%Tween 20). Primary antibodies used included rabbit polyclonalanti-caspase-3 (Santa Cruz Biotechnology, H-277, Santa Cruz, Calif.),goat polyclonal anti-actin (Santa Cruz Biotechnology, C-11), rabbitpolyclonal anti-MnSOD (Stressgen, SOD-111, Victoria, BC, Canada), mousemonoclonal anti-bcl-2 (Santa Cruz Biotechnology, 100),), mousemonoclonal anti-bcl-x_(L) (H-5), and mouse monoclonal anti-bax (SantaCruz Biotechnology, B-9). The anti-rabbit, goat, and mouse secondaryantibodies were conjugated to horseradish peroxidase and detected withECL western blotting detection reagents (Amersham Biosciences,Piscataway, N.J.).

Determination of Intracellular GSH and GSSG Content.

The determination of intracellular GSH and GSSG content followingexposure to selenite was performed using the GSH-reductase recyclingassay (Vandeputte et al., Cell Biol Toxicol, 10: 415-421 (1994)). Thisassay measures the reaction of GSH with 5,5′-dithiobis(2-nitrobenzoicacid) (DTNB). Briefly, cells (floating and adherent) were rinsed in coldstock buffer (143 mM sodium phosphate, 6.3 mM EDTA, pH7.4), and lysed byrepeated freeze thawing in 130 μl of 10 mM HCl. Following lysis a 10 μlsample was removed and used for protein determination. Proteins wereprecipitated on ice for 15 min with 30 μl of 6.5% sulfosalicyclic acidand centrifuged for 15 min at 2000×g and 4° C. The supernatants werecollected and stored at −80° C. until assayed. The 96-well plate assaywas prepared by mixing 20 μl of sample or known GSH standard, 20 μl ofstock buffer, 200 μl of reaction buffer (1 mM5,5′-dithiobis(2-nitrobenzoic acid (DTNB) and 0.34 mM nicotinamideadenine dinucleotide phosphate (NADPH) in stock buffer), and 40 μl ofGSH reductase (8.5 U/ml). The reaction was recorded kinetically at30-sec intervals for 6 min at a wavelength of 405 nm GSSG was measuredfollowing GSH derivatization with 2-vinylpyridine and triethanolamine.GSSG was measured as described above using known GSSG standards. Theconcentration of GSH and GSSG was expressed as nmol/mg of protein.

Clonogenic Survival Assays.

In Examples 1-4 below, LAPC-4 cells were treated with or withoutselenite for 6 hours prior to receiving 2 Gy of ionizing radiation. Inother Examples, LAPC-4 and DU 145 cells were treated with 10 μM selenitefor 6 or 12 hours treatment with 2 or 5 Gy of γ-irradiation.

Following irradiation cells were trypsinized, counted, and seeded intriplicate into 60 mm dishes. At least two dilutions of cells were usedfor each treatment group. Plated cells were allowed to grow for 14 or 17days before being stained with 0.25% crystal violet in 75% ethanol.Resulting colonies with greater than 50 cells were scored. The survivingfraction was calculated as the plating efficiency of treated cellsdivided by the plating efficiency of untreated cells. SF₂ is thesurvival fraction of exponentially growing cells that were irradiated atthe clinically relevant dose of 2 Gy. The SF₂ enhancement ratio (SF₂ ER)is defined here as the SF₂ without treatment divided by the SF₂ forcells treated with selenite.

Example 1 Selenite Effects on Apoptosis in LAPC-4 Prostate Cancer Cellsand Primary Prostate (Normal and Cancer) Cell Strains and the GSH:GSSGRatio as a Measure of Apoptotic Potential

LAPC-4 cells were chosen for these studies because they share manybiologically relevant features with newly diagnosed prostate cancer inpatients (e.g. expression of prostate specific antigen (PSA), androgenreceptor (AR), prostatic acid phosphatase and wild-type p53) (Sawyers etal., U.S. Pat. No. 6,365,797). Selenite induced apoptosis in adose-dependent fashion in LAPC-4 prostate cancer cells at 48 hours invitro. Apoptosis was measured with the DNA binding dye propidium iodide,which measures cells in the sub-G1 fraction of the cell cycle as theyundergo apoptosis.

LAPC-4 cells pre-treated with 500 μM BSO for 24 hours, showed decreasedselenite-induced apoptosis, suggesting GSH acts as a selenite cofactorthat facilitates cell death (FIG. 2, Panel A). Selenite treated LAPC-4cells also showed a significant increase in cleaved caspase 3 (FIG. 2,Panel B). The induction of apoptosis by selenite in LAPC-4 cells wasassociated with a decrease in the intracellular ratio of GSH:GSSG aswell as the total GSH (FIG. 2, Panels C and D). Interestingly, treatmentof these cells with BSO (500 μM for 72 hrs), which produced a similardecrease in the intracellular GSH content, did not induce significantapoptosis. Therefore, other factors in addition to GSH depletion, suchas superoxide production and altered redox signaling as a result ofselenite metabolism, appear to contribute to selenite-induced apoptosis.Depletion of GSH alone does not explain or predict the effects ofselenite, nor does the ability of a compound to deplete GSH-predict thatthe compound will have the effects of selenite.

Primary cultures of epithelial cells allow for comparative studiesbetween normal and cancer cells derived from the same subject andcultured under identical conditions (Peehl, 2002). As short-termcultures, primary cultures may also be more realistic models of thebehavior of prostate cancer than established cell lines. In experimentsin which matched primary cell strains were used, normal prostate cells(from the peripheral zone (PZ), where the majority of prostaticadenocarcinomas originate) were resistant to selenite-induced apoptosiscompared to the corresponding prostate cancer derived cells (CA) at 48hours (FIG. 3, Panels A, B, and C). Normal prostate and prostate cancercell strains have a similar growth rate in culture (FIG. 3, Panel D),therefore, the differential response to selenite does not appear to bethe result of differences in their rate of proliferation.

Prostate cancer cells were found to have higher basal intracellularGSH:GSSG ratios than those cells derived from normal tissue. Total GSHconcentrations were similar for each pair of normal and cancer cells,however, there were differences in total GSH between the three differentmatched pairs (FIG. 4, Panels A, B, and C). The increased ratio ofGSH:GSSG in cancer cells was due to the fact that they had significantlyless GSSG than the corresponding normal cells. Following treatment withselenite, prostate cancer cells (E-CA-3) showed a greater reduction inthe ratio of GSH:GSSG compared to normal cells (E-PZ-3) (FIG. 5, PanelsA and B). For example, the GSH:GSSG ratio was decreased 41.1% in normalcancer cells after being exposed to 25 μM selenite for 48 hours, whereasthe ratio was decreased 65.9% in the cancer cells. Therefore, seleniteappears to have a greater effect upon the intracellular redox state ofprostate cancer cells as compared to normal cells. Based upon theseresults, there appears to be a direct relationship between the level ofreduced GSH in prostate cells and the potency of selenite-inducedcytotoxicity. The rank order of sensitivity to selenite from lowest tohighest, which coincided with intracellular GSH status, was normal humanprostate, primary carcinoma, and LAPC-4 prostate cancer cells.

In order to assess the intracellular redox state over time, total GSHand GSSG concentrations were measured at 6, 24, and 48 hours aftertreatment with 10 μM selenite. Selenite decreased total GSH levels in atime-dependent fashion from a basal level of 52.1±5.6 nmol/mg to11.8±2.1 nmol/mg at 48 hours (FIG. 6, Panel A). The concentration ofGSSG increased following selenite treatment and was maximal after 24hours (FIG. 6, Panel B). As a result, the ratio of GSH:GSSG in LAPC-4cells was decreased by selenite (FIG. 6, Panel C). As early as 6 hoursafter treatment with 10 μM selenite, the GSH:GSSG ratio was decreasedfrom 129.4±13.6 to 15.1±2.3. These changes in intracellular GSH contentin response to selenite indicate a dramatic shift in the cellular redoxbalance towards an oxidative state.

Example 2 Selenite-Induced Apoptosis in LAPC-4 Prostate Cancer Cells isDose-Dependent

The proliferation of LAPC-4 cells was measured using the MTS cellularproliferation assay after incubation with selenite for 48 hours. Cellproliferation was 53.3% of control after treatment with 10 μM selenite,and 33.4% of control after treatment with 25 μM selenite (FIG. 7, PanelA). Apoptosis was measured as the percentage of cells in the sub-G1fraction of the cell cycle. The percentage of sub-G1 cells followingtreatment with 10 or 25 μM selenite for 48 hours was 14.5% and 26.1%,respectively (FIG. 7, Panel B). Cleavage of caspase-3, a marker ofapoptosis, was also detected in selenite-treated LAPC-4 cells by Westernblotting (FIG. 7, Panel C). In contrast, primary cultures of normalprostate epithelial cells were more resistant to selenite-inducedapoptosis than LAPC-4 cells (FIG. 7, Panel D). These data show thatselenite inhibited cell growth and induced apoptosis in a dose-dependentfashion in androgen-dependent LACP-4 human prostate cancer cells invitro.

Example 3 MnSOD Protein Expression in Primary Prostate Cell Strains

Mangenese superoxide dismutase (MnSOD) is an anti-oxidant enzyme locatedin the mitochondrial matrix that scavenges superoxide anions produced asa consequence of aerobic respiration (Pani et al., Cancer Res,60:4654-60 (2000)). Since superoxide is a by-product of selenitemetabolism, MnSOD protein expression was measured in normal and cancercell strains.

Normal prostate cells had significantly higher basal levels of MnSODexpression than the prostate cancer cells. After selenite treatment for48 hours the normal cells showed little or no induction of MnSODprotein, whereas in the prostate cancer cells (E-CA-1 and E-CA-2),selenite induced MnSOD protein expression in a dose-dependent fashion(FIG. 8). MnSOD expression is highly inducible by various agents andconditions that cause oxidative stress, which suggests that more ROS isgenerated in prostate cancer cells in response to Selenite than innormal cells. The difference in basal MnSOD expression may contribute tothe differential effects of Selenite in prostate cancer versus normalcells. Relate expression back to original tissues from which they werederived.

Example 4 Effects of Selenite on Bcl-2 and Bax Expression in NormalProstate and Cancer Primary Cell Strains

The Bcl-2 protein family is among many key regulators of apoptosis.Anti-apoptotic Bcl-2 associates with the outer mitochondrial membrane toregulate the mitochondrial membrane potential, counteract the effect ofthe pro-apoptotic Bax, and block the release of cytochrome c, which isimportant for apoptotic signaling (Adams et al., Science, 281: 1322-1326(1998)). Dynamic changes in the Bcl-2:Bax ratio appear to be importantin the induction of apoptosis in prostate cancer cells.

Selenite (48 hours) decreased the Bcl-2:Bax expression ratio in LAPC-4and primary prostate cancer derived cells (FIG. 9, Panels A and B)Whether the decrease in the Bcl-2:Bax ratio is the result of decreasedintracellular GSH and/or increased ROS is not known. In contrast, normalprostate cells, which are more resistant to the effects of selenite,show no change or increased Bcl-2 protein expression and no change orslight induction of Bax-α expression in response to selenite.

Expression levels of bcl-2, bcl-x_(L), and bax were also assessed afterexposure to 10 μM and 25 μM selenite selenite for 48 hours by Westernblotting (FIG. 10). The expression of anti-apoptotic, bcl-2 andbcl-x_(L), were decreased following treatment with selenite, and thisreduction was coupled to an increased expression of pro-apoptotic bax.The decreased bcl-2:bax expression ratio indicates that selenite-inducedapoptosis in LAPC-4 cells correlates with a shift in the balance ofBcl-2 family member expression from a pro-survival to apoptotic state.

Example 5 Selenite Sensitizes LAPC-4 Cells to Radiation-Induced CellKilling

Prostate cancer patients with elevated Bcl-2:Bax ratios are at increasedrisk of radiation therapy failure (Mackey et al., Urology, 52(6):1085-1090 (1998)). Since the above examples showed that selenite candecrease the GSH:GSSG and Bcl-2:Bax ratios in LAPC-4 cells, the abilityof Selenite to sensitize LAPC-4 cells to γ-irradiation was tested.LAPC-4 cells exposed to 10 μM or 25 μM selenite for 6 hours prior toreceiving 2 Gy of ionizing radiation showed significantly decreasedsurvival compared to cells treated with selenite or irradiation alone asmeasured by clonogenic survival assay (FIG. 11.

In order to assess the effects of higher doses of γ-radiation, LAPC-4cells were treated with 10 μM selenite for 6 hours prior to receiving 2Gy or 5 Gy γ-irradiation. Survival was measured using a clonogenicassay. This treatment regimen was based upon the data above showing thattreatment of LAPC-4 cells with 10 μM selenite for 6 hours decreased theGSH:GSSG ratio 88.3%. The surviving fraction of LAPC-4 cells aftertreatment with selenite alone was 0.431±0.021 (data not shown). Inexperiments, in which selenite was combined with radiation, the resultswere normalized for the killing from selenite alone. Selenite enhancedradiation-induced inhibition of colony formation (SF₂=0.056) compared tocells treated with radiation alone (SF₂=0.244) (FIG. 12). These resultsindicate that selenite inhibits the clonal growth of LAPC-4 cells andenhances the effect of radiation on these cells.

Example 6 Radiosensitization of Androgen-Independent DU 145 Cells bySelenite

An androgen-independent prostate cancer cell line was examined todetermine if selenite would similarly sensitize these cells toγ-irradiation. The androgen-independent DU 145 prostate cancer cell linewas chosen because previous studies have shown that selenite can inhibitgrowth and induce apoptosis in these cells (Shen et al., 1999 Int. J.Cancer. 81: 820-828). Again, changes in intracellular GSH and GSSG weremeasured 6 and 12 hours after treatment with 10 μM selenite. The ratioof GSH:GSSG decreased from a basal level of 146.4±14.0 in controls cellsto 57.5±13.8 and 5.7±1.8 at 6 and 12 hours, respectively (FIG. 13, PanelA).

The effects of selenite on the response of DU 145 cells to γ-irradiationwere studied using clonogenic survival assays. The surviving fractionsof DU 145 cells treated with 10 μM selenite for 6 or 12 hours alone (andassessed at 17 days after irradiation) were 0.941 and 0.409,respectively (data not shown). After normalization for the killing fromselenite alone, pre-treatment with selenite enhanced radiation-inducedcell death (SF₂=0.343 and 0.199 at 6 and 12 hours, respectively)compared to cells treated with radiation alone (SF₂=0.554) (FIG. 13,Panel B). These results are summarized in Table 1.

TABLE 1 Radiosensitization of LAPC-4 and DU 145 cells by selenite CellLine Treatment SF₂ SF₂ ER LAPC-4 Radiaton alone 0.244 10 μM Selenite 6hr pre Radiation 0.056 4.36 DU 145 Radiaton alone 0.554 10 μM Selenite 6hr pre Radiation 0.343 1.62 10 μM Selenite 12 hr pre Radiation 0.1992.78

These data indicate that the iSe and radiation combination therapy ofthe invention can be useful in enhancing radiosensitivity ofhormone-dependent and hormone-independent cancers. Moreover, thecombination therapy of the invention can be used to treat cancers havingwild-type or mutant tumor suppressor genes. LAPC-4 cells have wild-typep53 and Rb tumor suppressor genes, while DU 145 cells contain mutant p53and Rb.

Example 7 Analysis of Effect of Inorganic Selenite In Vivo in aNon-Human Animal Model

In vivo studies are performed in scid mice with subcutaneous LAPC-4xenograft prostate cancer tumors on the upper leg/flank. Mice areinjected with 1×10⁶ cells suspended in matrigel. Once the tumors havereached approximately 100-150 mm³ in size, mice are treated with 1, 2 or4 mg/kg Selenite in phosphate buffered saline by subcutaneous injectionthree times per week (with 7 mice per group) as described in previousstudies (Milner et al., Fed Proc, 44: 2568-2572 (1985); Combs et al.,Selenium and cancer. In: Antioxidants and Disease Prevention, Ch. 8,97-113. CRC Press, N.Y. (1997); Shamberger et al., CRC Crit Rev ClinSci, 2: 211-219 (1971)).

The length, width, and height (mm) of the tumors are measured withcalipers before treatment and three times a week thereafter until thetumor volume is at least (4×) the original pretreatment volume. Theanimals are sacrificed when the tumors reach the predetermined size.Tumor volume (mm³) are calculated according to the formula: tumorvolume=π/6×length×width×height. The data are expressed as percent of thepretreatment volume on day 0, or as the mean tumor volume quadruplingtime (TVQT, in days)±standard error, and the tumor growth delay (TGD, indays) time. The TGD time of each group are expressed as the differencebetween the TVQT of treated tumors compared to that of untreated controltumors. Data are analyzed using a two-tailed Student's t-test. Animalbody weight is also recorded as an indicator of toxicity. 2 mice pergroup are sacrificed at 48 and 168 hours (2 and 7 days) followingtreatment for measurement of apoptosis and GSH content. In the event ofsignificant tumor shrinkage, tumors are excised at earlier time points.Apoptosis in treated tumors are quantitated by TdT-mediated dUTP nickend labeling (TUNEL) staining of fixed tissue sections using fluorescentmicroscopy (53). The GSH content within the tumor will also be measuredusing a GSH-reductase recycling assay.

Example 8 Analysis of Effect of Inorganic Selenite and RadiationCombination Therapy In Vivo in a Non-Human Animal Model

A pilot dose finding experiment is performed with 5 mice per grouptreated with 0, 5, 10, and 15 Gy (local radiation) given as a singlefraction to the tumor. Mice are irradiated with a Philips 200 kVp X-rayunit (12.5 mA; half-value layer, 1.0 Cu), at a dose rate ofapproximately 1.0 Gy/min, in round plexiglass jigs with custom leadblock shielding to limit the radiation therapy field to the tumor withmargin. A dose that results in measurable tumor shrinkage but not a cureare selected for further study.

For Selenite plus radiation therapy studies, 2 sets of experiments areconducted using A) radiation given as a single dose (as identifiedabove) or B) radiation given in multiple doses (3 Gy per day for 5days). The first experiments will consist of the following groups: A)untreated control, B) Selenite alone (using a dose regime previouslydemonstrated to cause the greatest but non-curative reduction in tumorgrowth from Example 5), C) radiation alone (single dose determined frompilot study) and D) Selenite plus radiation. The second set ofexperiments include the following groups: A) untreated control, B)Selenite alone, C) radiation alone (fractionated schedule of 3 Gy perday for 5 days) and D) Selenite plus fractionated radiation therapy.Each treatment group contains 10 mice. The same schedule as in Example 6is used for sacrifice and measurement of specified endpoints. Tumorresponse data are analyzed as described in Example 6.

Example 9 Evaluation of Therapeutic Index of Selenite CombinationTherapy

A net gain in the therapeutic index using selenite as a potentialradiosensitizer or chemotherapy sensitizer for the treatment ofneoplastic disease requires that selenite not have a similarradiosensitizing effect in normal tissues. The mucosa of thegastrointestinal tract is one of the most radiosensitive tissues in theabdominal/pelvic area. Analysis of effects on mucosa is particularlyclinically relevant for the treatment of prostate cancer with radiation,since diarrhea and rectal problems are not uncommon side effects ofradiation therapy to the prostate (and regional lymphatics).

The effects of Selenite combined with radiation therapy ongastrointestinal mucosa are assessed using the well characterized andvalidated intestinal crypt cell survival assay (Franker et al., MethodsCell Biol, 46: 57-76 (1995); Peehl et al., In Vitro, 24: 526-530(1988)). C3H/SPF mice are used for this analysis, because unlike scid ornude mice, they are immunologically normal and do not have an inherentdefect in DNA repair (like scid mice) that would increase theradiosensitivity of normal tissues. The inventors have extensiveexperience with this assay in this strain of mouse and have previouslydemonstrated that 12 Gy as a single dose will induce sufficientradiation damage to allow for the detection of either protection orenhanced damage by combining a radiation modulating agent with radiationtherapy (Peehl, 1988).

This study has 4 experimental groups: Untreated control, 12 Gyabdominal/pelvis radiation therapy alone, Selenite (at the optimalradiosensitizing dose and schedule identified in the experiments above)and 12 Gy radiation+Selenite. There are 5 mice per group. Male C3H/SPFmice are irradiated with a Philips 200 kVp X-ray unit (as describedabove) in round plexiglass jigs with custom lead block shielding tolimit the radiation therapy field to the abdomen and pelvis. Mice aresacrificed 90 hours after irradiation and the survival of intestinalcrypt stem cells are assessed by the microcolonoy method as previouslydescribed (Franker, 1995; Peehl, 1988). This method, including thescoring method and statistical method utilized for analyzing this datahas been described in detail (Peehl, 1988).

Example 10 Effect of Selenite and Radiation Combination Therapy In Vivoin a Non-Human Animal Model

Mice with well-established LAPC-4 tumors were treated with selenitealone, local tumor radiation alone, or selenite with localized tumorradiation. selenite significantly enhanced local radiation-induced tumorgrowth delay. The effect of the combined treatment was significantlygreater than that of radiation or selenite alone. Furthermore, selenitetreatment was very well tolerated, and there was no significant weightloss in the selenite treated mice compared to the group of mice treatedwith local tumor irradiation alone.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method of treating a neoplastic disease in a subject, the methodcomprising: administering to a subject having a tumor a pharmaceuticallyacceptable salt of an inorganic selenium-containing compound (iSecompound) in an amount effective to alter a reduction-oxidation state ofa tumor cell toward oxidation; and administering a cancer therapy otherthan an iSe compound to the subject; wherein the neoplastic disease inthe subject is treated.
 2. The method of claim 1, wherein the iSecompound is inorganic selenite.
 3. The method of claim 1, wherein thecancer therapy is administered after administering the iSe compound. 4.The method of claim 1, wherein the neoplastic disease is prostatecancer.
 5. The method of claim 1, wherein the cancer therapy isradiation therapy.
 6. The method of claim 5, wherein the radiationtherapy is external beam radiation therapy, brachytherapy orsystemically targeted radiation.
 7. The method of claim 1, wherein thecancer therapy is a chemotherapeutic agent.
 8. The method of claim 1,wherein said administering of the iSe compound is oral, intravenous,tumor targeted, intratumoral, or peritumoral.
 9. The method of claim 1,wherein the iSe compound is administered for a time sufficient formetabolism of the iSe compound prior to administering the cancertherapy.
 10. A method of enhancing sensitivity of a tumor in a subjectto a cancer therapy, the method comprising: administering to a subjecthaving a tumor a pharmaceutically acceptable salt of an inorganicselenium-containing (iSe) compound in an amount effective to sensitizethe tumor to a cancer therapy; and administering the cancer therapy tothe subject; wherein administration of the iSe compound is effective toenhance sensitivity of the tumor to the cancer therapy.
 11. The methodof claim 10, wherein inorganic selenium-containing compound is inorganicselenite.
 12. The method of claim 11, wherein the cancer therapy is areactive oxygen species (ROS)-inducing therapy.
 13. The method of claim12, wherein the ROS-inducing cancer therapy is a radiotherapy.
 14. Themethod of claim 12, wherein the ROS-inducing cancer therapy is achemotherapeutic drug or a biological response modifier.
 15. A method oftreating prostate cancer, the method comprising: administering to asubject having prostate cancer a pharmaceutically acceptable salt of aninorganic selenite; and administering a cancer therapy other thaninorganic selenite to the subject; wherein administering the inorganicselenite and the cancer therapy provides for a synergistic effect inprostate cancer cell growth inhibition to treat the prostate cancer. 16.The method of claim 15, wherein the cancer therapy is a reactive oxygenspecies (ROS)-inducing therapy.
 17. The method of claim 16, wherein theROS-inducing cancer therapy is a radiotherapy.
 18. The method of claim16, wherein the ROS-inducing cancer therapy is a chemotherapeutic drugor a biological response modifier.
 19. The method of claim 17, whereinthe radiotherapy comprises external radiation therapy, brachytherapy(internal radiation therapy), or systemically targeted radiation. 20.The method of claim 13, wherein the radiotherapy comprises externalradiation therapy, brachytherapy (internal radiation therapy), orsystemically targeted radiation.